Method of rna in vitro transcription using a buffer containing a dicarboxylic acid or tricarboxylic acid or a salt thereof

ABSTRACT

The present invention relates to a buffer system comprising a dicarboxylic acid or tricarboxylic acid or a salt thereof for synthesizing RNA molecules as well as a method of RNA in vitro transcription using this buffer system. The present invention also provides the use of this buffer system in RNA in vitro transcription and in the reduction or prevention of precipitates during RNA in vitro transcription.

This application is a continuation of U.S. application Ser. No.16/012,751, filed Jun. 19, 2018, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No.PCT/EP2016/082534, filed Dec. 23, 2016, which claims benefit ofInternational Application No. PCT/EP2015/081175, filed Dec. 23, 2015,the entire contents of each of which are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to a buffer system comprising adicarboxylic acid or tricarboxylic acid or a salt thereof forsynthesizing RNA molecules as well as a method of RNA in vitrotranscription using this buffer system. The present invention alsoprovides the use of this buffer system in an RNA in vitro transcriptionprocess and in the reduction or prevention of precipitates during RNA invitro transcription.

INTRODUCTION

Therapeutic ribonucleic acid (RNA) molecules represent an emerging classof drugs. RNA-based therapeutics include mRNA molecules encodingantigens for use as vaccines (Fotin-Mleczek et al. (2012) J. Gene Med.14(6):428-439). In addition, it is envisioned to use RNA molecules forreplacement therapies, e.g. providing missing proteins such as growthfactors or enzymes to patients. Furthermore, the therapeutic use ofnon-coding immunostimulatory RNA molecules and other non-coding RNAssuch as microRNAs, siRNAs, CRISPR/Cas9 guide RNAs, and long non-codingRNAs is considered.

For the successful development of RNA therapeutics, the production ofRNA molecules as active pharmaceutical ingredients must be efficient interms of yield, quality, safety and costs, especially when RNA isproduced at a large scale.

In the art, straight-forward processes for the recombinant production ofRNA molecules in preparative amounts have been developed in a processcalled “RNA in vitro transcription”. The term “RNA in vitrotranscription” relates to a process wherein RNA is synthesized in acell-free system (in vitro). RNA is commonly obtained by enzymatic DNAdependent in vitro transcription of an appropriate DNA template, whichis often a linearized plasmid DNA template. The promoter for controllingRNA in vitro transcription can be any promoter for any DNA dependent RNApolymerase. Particular examples of DNA dependent RNA polymerases are thebacteriophage enzymes T7, T3, and SP6 RNA polymerases.

Methods for RNA in vitro transcription are known in the art (see forexample Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle etal. (2013) Methods Enzymol. 530: 101-14). Reagents used in said methodsmay include: a linear DNA template with a promoter sequence that has ahigh binding affinity for its respective RNA polymerase; ribonucleosidetriphosphates (NTPs) for the four bases (adenine, cytosine, guanine anduracil); a cap analog (e.g., m7G(5′)ppp(5′)G (m7G)); other modifiednucleotides; DNA-dependent RNA polymerase (e.g., T7, T3 or SP6 RNApolymerase); ribonuclease (RNase) inhibitor to inactivate anycontaminating RNase; pyrophosphatase to degrade pyrophosphate, whichinhibits transcription; MgCl₂, which supplies Mg²⁺ as a cofactor for theRNA polymerase; antioxidants (e.g. DTT); polyamines such as spermidine;and a buffer to maintain a suitable pH value.

Common buffer systems used in RNA in vitro transcription include4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) andtris(hydroxymethyl)aminomethane (Tris). The pH value of the buffer iscommonly adjusted to a pH value of 6 to 8.5. Some commonly usedtranscription buffers comprise 80 mM HEPES/KOH, pH 7.5 and 40 mMTris/HCl, pH 7.5.

The transcription buffer also contains a magnesium salt such as MgCl₂commonly in a range between 5-50 mM. Magnesium ions (Mg²⁺) are anessential component in an RNA in vitro transcription buffer systembecause free Mg²⁺ acts as cofactor in the catalytic center of the RNApolymerase and is critical for the RNA polymerization reaction. Indiffuse binding, fully hydrated Mg ions also interact with the RNAproduct via nonspecific long-range electrostatic interactions.

RNA in vitro transcription reactions are typically performed as batchreactions in which all components are combined and then incubated toallow the synthesis of RNA molecules until the reaction terminates. Inaddition, fed-batch reactions were developed to increase the efficiencyof the RNA in vitro transcription reaction (Kern et al. (1997)Biotechnol. Prog. 13: 747-756; Kern et al. (1999) Biotechnol. Prog. 15:174-184). In a fed-batch system, all components are combined, but thenadditional amounts of some of the reagents are added over time (e.g.,NTPs, MgCl₂) to maintain constant reaction conditions.

Moreover, the use of a bioreactor (transcription reactor) for thesynthesis of RNA molecules by in vitro transcription has been reported(WO 95/08626). The bioreactor is configured such that reactants aredelivered via a feed line to the reactor core and RNA products areremoved by passing through an ultrafiltration membrane (having a nominalmolecular weight cut-off, e.g., 100,000 daltons) to the exit stream.

Problem in the Art

As discussed above, magnesium ions (Mg²⁺) are an essential component inan RNA in vitro transcription buffer system because free Mg²⁺ acts ascofactor in the catalytic center of the RNA polymerase and is thereforecritical for the RNA polymerization reaction. Conventional buffersystems for RNA in vitro transcription (e.g., HEPES buffer, Tris-HClbuffer) contain high concentrations of free magnesium ions, because freeMg²⁺ ions are required to guarantee a high activity of the RNApolymerase enzyme. However the free Mg²⁺ ions present in buffer systemsknown in the art can cause dramatic problems, especially in the contextof high-yield/industrial-scale RNA production. Some of the majorproblems associated with free Mg²⁺ ions in the production of RNA areoutlined below.

First, the binding of magnesium to RNA can cause integrity problems,because Mg²⁺ ions can catalyze self-cleavage of the RNA product, leadingto undesired abortive sequences (Forconi, Marcello, and Daniel Herschlag(2009) Methods in enzymology 468: 91-106).

Second, during RNA in vitro transcription, the enzymatic polymerizationreaction generates one mole pyrophosphate per mole incorporatednucleotide triphosphate (NTP): (NMP)_(n)+NTP→(NMP)_(n+1)+PP_(i). Thepyrophosphate (PP_(i)) is then cleaved into two moles ortho phosphate bythe enzyme pyrophosphatase (P₂O₇ ⁴⁻+H₂O→2HPO₄ ²⁻). However, thehydrolysis of PPi to Pi during the running reaction is not sufficient toovercome turbidity of the reaction as widely believed in the art. Freemagnesium and phosphate ions result in the precipitation of magnesiumphosphate salts, too (e.g., Mg(H₂PO₄)₂; MgHPO₄; Mg₃(PO₄)₂). In additionto that, hydrated magnesium ions interact with the RNA product vianonspecific long-range electrostatic interactions, which may lead to amagnesium-driven precipitation of the RNA product.

The generation of magnesium-driven precipitates as outlined above cancause major problems in the production process of RNA, especially inlarge-scale/industrial-scale RNA production:

First of all, precipitations as described above will have an impact onRNA production costs because RNA product, DNA template, enzyme (RNApolymerase, pyrophosphatase, RNAse inhibitors) and raw materials (NTPs,cap analog) may co-precipitate with magnesium-dependent precipitates.

Next, precipitation introduces uncontrolled changes in the reactionconditions (e.g., NTP concentration, enzyme concentration, bufferconditions), which has to be avoided in a robust process applicable forindustrial RNA production.

Magnesium-driven precipitation may lead to a drop in the free Mg²⁺concentration, resulting in depletion of magnesium ions from the RNApolymerase reaction center. A consequence of that would be a lessefficient RNA in vitro transcription.

In addition, uncontrolled reaction conditions, caused by themagnesium-driven precipitation, are highly problematic in the context offeeding strategies, because the feeding of raw materials (e.g., NTPs)cannot be precisely calculated and adjusted any more. In addition tothat, in the context of continuous RNA production processes, anaccumulation of precipitate over time may occur, which can eventuallylead to a stop of the production process.

Quantification of nucleotides is commonly performed using UV-lightspectrometry. In a RNA transcription reaction it is beneficial toquantify free NTPs using spectrometry. With such a measurement, theprogress of RNA in vitro transcription can be calculated because thedecrease of free NTPs can be translated back into the produced RNAmolecules. Such a measurement is technically applicable in settingswhere the transcription buffer (containing NTPs) is separated from theRNA product and the DNA template (e.g., via a low molecular weightcutoff membrane). In such a setting, magnesium-driven precipitationwould cause a turbidity of the buffer solution and that, in consequence,would impair spectrometric quantification of NTPs.

Moreover, precipitation as described above may cause general proceduralproblems because e.g., filtration membranes, tubing systems and otherelements of a transcription setting (e.g., transcription reactor) may beclogged by the precipitates.

Magnesium-driven precipitates may also cause problems during thepurification procedure of the RNA product. For example, a directpurification of the RNA product from the RNA in vitro transcriptionreaction by methods such as tangential flow filtration may be largelyimpeded by precipitates.

Finally, a depletion of ions caused by precipitations may cause furtherproblems downstream of the RNA in vitro transcription reaction, becausethe hydrolysis of the DNA template via DNAses (which conventionallyhappens in the IVT buffer after addition of CaCl₂) may be impeded whichmay eventually lead to DNA contaminations in the final RNA product.

In view of the above described problems, there is a continued need forimproved conditions for the RNA in vitro transcription reaction,especially in the establishment of robust large-scale RNA productionsettings. Therefore, preventing magnesium-driven precipitation asdescribed above would be a major advantage in the art, and would improvethe establishment of a robust and cost-effective industrial RNAproduction process.

Solution of the Problem and Description of the Invention

The problems outlined above are solved by the subject-matter of thepresent invention.

In an initial experiment, the inventors characterized the precipitatesformed during enzymatic RNA in vitro transcription using astate-of-the-art HEPES buffer. They could show that magnesium-drivenprecipitates contain RNA and proteins (e.g., RNA polymerase, RNAseInhibitor) (see FIG. 1).

In a next set of experiments, the inventors showed that the use ofTris-citrate buffer surprisingly prevents the formation of precipitates(Tris pH 7.5; 24 mM citric acid) in the process of RNA in vitrotranscription. The effect was not observed with another organic acidsuch as acetate (see FIG. 2).

To further characterize the effect of citrate on the prevention ofprecipitation, an experiment was conducted where differentconcentrations of citrate (in a Tris buffer, pH 8.0) were used in an RNAin vitro transcription experiment, ranging from 0-25 mM citrate. Theresults showed that the reduction of precipitations was a dose-dependenteffect of citrate. Moreover, the experiment showed that at citrateconcentrations >10 mM, the formation of precipitates was alreadystrongly reduced, and at concentrations >15 mM, the formation ofprecipitates could be prevented (see FIG. 3).

To characterize whether the effect of citrate observed for Tris buffersis also transferrable to other buffers such as HEPES, the inventorsconducted an experiment where different concentrations of citrate,ranging from 0-25 mM citrate, were used in a HEPES buffer in an RNA invitro transcription experiment. The results showed that the reduction ofprecipitations was a dose-dependent effect of citrate in HEPES buffer.Moreover, the experiment showed that at citrate concentrations >10 mM,the formation of precipitates could be prevented (see FIG. 4).

Finally, an experiment was conducted to investigate the effect ofcitrate in the RNA in vitro transcription reaction on the subsequenthydrolysis of the DNA template by adding DNase1 and calcium chloride tothe RNA in vitro transcription reaction. The results showed that the useof citrate in the RNA in vitro transcription reaction promotes thehydrolysis of the DNA template (see FIG. 5).

Summarizing the above, the inventors surprisingly showed that by using atranscription buffer (Tris, HEPES) containing citrate, precipitationscould be prevented during RNA in vitro transcription and theDNase-mediated hydrolysis of the DNA template could be improved.

Citrate containing buffer systems as disclosed in the present inventionare broadly applicable for RNA in vitro transcription, solving theproblems associated with magnesium-driven precipitations, especially inthe context of industrial RNA production.

Accordingly, the present invention relates to a method for in vitrotranscription of a nucleic acid template into RNA, comprising the stepsof:

-   -   providing a mixture comprising a dicarboxylic or tricarboxylic        acid or salt thereof, a buffer substance, ribonucleoside        triphosphates, one or more magnesium salts, said nucleic acid        template and RNA polymerase; and    -   incubating the reaction mixture under suitable conditions.

The present invention also relates to a method for in vitrotranscription of a nucleic acid template into RNA, comprising the stepsof:

-   -   providing a mixture comprising a dicarboxylic or tricarboxylic        acid or salt thereof, a buffer substance, ribonucleoside        triphosphates, one or more magnesium salts, said nucleic acid        template and a recombinant RNA polymerase, wherein the mixture        does not comprise a proteinogenic amino acid or tRNA; and    -   incubating the reaction mixture under suitable conditions.

Additionally, the present invention relates to a method for preparingRNA, comprising the steps of:

a) incubating a mixture comprising a dicarboxylic or tricarboxylic acidor salt thereof, a buffer substance, ribonucleoside triphosphates, oneor more magnesium salts, a nucleic acid template and a recombinant RNApolymerase under suitable conditions;

b) adding a DNase to the mixture of a) and incubating the resultingmixture containing DNase under suitable conditions.

The dicarboxylic or tricarboxylic acid or salt thereof may be citricacid or a salt thereof.

In one embodiment, the concentration of citric acid or salt thereof isat least half of the concentration of magnesium ions present in the invitro transcription reaction.

The buffer substance may be Tris base, HEPES or Tris-HCl.

In one embodiment, the concentration of the buffer substance is 10 to100 mM.

The dicarboxylic or tricarboxylic acid or salt thereof may be used toadjust the pH of the mixture.

In one embodiment the reaction mixture comprises Tris-citrate orHEPES-KOH plus sodium citrate, ribonucleoside triphosphates, one or moremagnesium salts, said nucleic acid template and RNA polymerase.

The magnesium salt may be magnesium chloride and the concentration ofmagnesium chloride may be 1 to 100 mM.

The reaction mixture may have a pH of 6 to 8.5 or of 7.5 to 8.0.

The RNA polymerase may be T7 RNA polymerase.

In one embodiment the total concentration of ribonucleosidetriphosphates in the mixture is between 0.1 and 60 mM.

The nucleic acid template may be a linearized plasmid DNA template.

The reaction mixture may comprise one or more of ribonuclease inhibitor,pyrophosphatase, cap analog, one or more antioxidants and one or moreamines and/or polyamines.

The antioxidant is DTT and the concentration of DTT may be 1 to 50 mM.

The polyamine may be spermidine and the concentration of spermidine maybe 1 to 25 mM.

The method may further comprise a step (c) of purifying the RNA whichmay comprise HPLC using a porous reversed phase as stationary phaseand/or tangential flow filtration.

In another aspect, the present invention relates to a buffer for invitro transcription of RNA comprising a dicarboxylic or tricarboxylicacid or a salt thereof and a buffer substance.

The present invention also relates to a buffer for in vitrotranscription of RNA comprising a dicarboxylic or tricarboxylic acid ora salt thereof and Tris as a buffer substance.

The dicarboxylic or tricarboxylic acid or a salt thereof may be citricacid or citrate and the concentration of citric acid or citrate may beat least 1 mM.

The buffer substance is Tris or HEPES and the buffer may be Tris-citrateor HEPES-KOH plus sodium citrate.

The buffer may have a pH of 6 to 8.5.

In another aspect, the present invention relates to a reaction mixturefor in vitro transcription comprising said buffer and ribonucleosidetriphosphates, one or more magnesium salts, a nucleic acid template andRNA polymerase.

The reaction mixture may further comprise one or more of ribonucleaseinhibitor, pyrophosphatase, cap analog, one or more antioxidants and oneor more amines and/or polyamines.

In another aspect, the present invention relates to a kit for in vitrotranscription, comprising said buffer or said reaction mixture.

In another aspect, the present invention relates to a bioreactor for invitro transcription, comprising said buffer or said reaction mixture.

In still another aspect the present invention relates to the use of adicarboxylic or tricarboxylic acid or a salt thereof in a method of invitro transcription of RNA.

In still another aspect the present invention relates to the use of adicarboxylic or tricarboxylic acid or a salt thereof in the reduction orprevention of the formation of precipitates in a method of in vitrotranscription.

The method of in vitro transcription may be as defined above.

In still another aspect the present invention relates to the use of adicarboxylic or tricarboxylic acid or a salt thereof in promoting thehydrolysis of DNA by DNase.

The DNA hydrolysis may take place after RNA in vitro transcription.

In still another aspect the present invention relates to the use ofbetaine in the in vitro transcription of a nucleic acid template with ahigh percentage of guanosine and cytosine nucleotides.

The concentration of betaine may be 10 mM to 1 M.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a qualitative characterization of the precipitates formedduring a HEPES buffered RNA in vitro transcription reaction. The pictureshows the result of an SDS-polyacrylamide gel electrophoresis ofprecipitate (1) and supernatant (2) at different time points (0 h-4 h)during RNA in vitro transcription. M=marker lane. (see Example 2)

FIG. 2 shows an analysis of precipitate formation during RNA in vitrotranscription reaction, testing the Tris-citrate buffer according to thepresent invention in comparison to Tris-acetate buffer and buffers knownin the art (HEPES, Tris-HCl). The formation of precipitates was measuredover time, using light spectrometry (absorption at 500 nm). For adetailed description of the experiment see Example 4.

FIG. 3 shows an analysis of precipitate formation during RNA in vitrotranscription reaction, testing different citrate concentrations,ranging from 0 mM to 25 mM, in a Tris buffer system at pH 8.0. For adetailed description of the experiment, see Example 5.

FIG. 4 shows an analysis of precipitate formation during RNA in vitrotranscription reaction, testing different citrate concentrations,ranging from 0 mM to 25 mM, in a HEPES buffer system at pH 8.0 after 120minutes RNA in vitro transcription. For a detailed description of theexperiment, see Example 6.

FIG. 5 shows a qPCR analysis of residual pDNA template present in theRNA after DNA hydrolysis. Buffer conditions of the DNAse treatment areindicated. Black columns: 3 mM CaCl2 and 200 U/ml DNAse1; Grey columns:3 mM CaCl2 and 400/ml DNAse1. The copy number of pDNA in the RNA isexpressed as copies/μg RNA. For a detailed description of theexperiment, see Example 7.

DEFINITIONS

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned in these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

In vitro transcription: The terms “in vitro transcription” or “RNA invitro transcription” relate to a process wherein RNA is synthesized in acell-free system (in vitro). DNA, particularly plasmid DNA, is used astemplate for the generation of RNA transcripts. RNA may be obtained byDNA-dependent in vitro transcription of an appropriate DNA template,which according to the present invention is preferably a linearizedplasmid DNA template. The promoter for controlling in vitrotranscription can be any promoter for any DNA-dependent RNA polymerase.Particular examples of DNA-dependent RNA polymerases are the T7, T3, andSP6 RNA polymerases. A DNA template for in vitro RNA transcription maybe obtained by cloning of a nucleic acid, in particular cDNAcorresponding to the respective RNA to be in vitro transcribed, andintroducing it into an appropriate vector for in vitro transcription,for example into plasmid DNA. In a preferred embodiment of the presentinvention the DNA template is linearized with a suitable restrictionenzyme, before it is transcribed in vitro. The cDNA may be obtained byreverse transcription of mRNA or chemical synthesis. Moreover, the DNAtemplate for in vitro RNA synthesis may also be obtained by genesynthesis.

Methods for in vitro transcription are known in the art (Geall et al.(2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) MethodsEnzymol. 530:101-14). Reagents used in said method typically include:

1) a linearized DNA template with a promoter sequence that has a highbinding affinity for its respective RNA polymerase such asbacteriophage-encoded RNA polymerases;

2) ribonucleoside triphosphates (NTPs) for the four bases (adenine,cytosine, guanine and uracil);

3) optionally a cap analog as defined below (e.g. m7G(5′)ppp(5′)G(m7G));

4) a DNA-dependent RNA polymerase capable of binding to the promotersequence within the linearized DNA template (e.g. T7, T3 or SP6 RNApolymerase);

5) optionally a ribonuclease (RNase) inhibitor to inactivate anycontaminating RNase;

6) optionally a pyrophosphatase to degrade pyrophosphate, which mayinhibit transcription;

7) MgCl₂, which supplies Mg²⁺ ions as a co-factor for the polymerase;

8) a buffer to maintain a suitable pH value, which can also containantioxidants (e.g. DTT), amines such as betaine and/or polyamines suchas spermidine at optimal concentrations.

In the method of RNA in vitro transcription according to the inventionno reagents which are only required for the in vitro translation of thetranscribed RNA to protein, but not for RNA in vitro transcription areused. In particular, the mixture used for RNA in vitro transcriptiondoes not contain any proteinogenic amino acid or tRNA. Further, themixture does not contain any proteinogenic amino acid, tRNA or a cellextract containing ribosomes.

Nucleic acid: The term nucleic acid means any DNA- or RNA-molecule andis used synonymous with polynucleotide. Furthermore, modifications orderivatives of the nucleic acid as defined herein are explicitlyincluded in the general term “nucleic acid”. For example, peptidenucleic acid (PNA) is also included in the term “nucleic acid”.

Nucleic acid template: The nucleic acid template provides the nucleicacid sequence which is transcribed into the RNA by the process of invitro transcription and which therefore comprises a nucleic acidsequence which is complementary to the RNA sequence which is transcribedtherefrom. In addition to the nucleic acid sequence which is transcribedinto the RNA the nucleic acid template comprises a promoter to which theRNA polymerase used in the in vitro transcription process binds withhigh affinity.

Preferably, the nucleic acid template may be a linearized plasmid DNAtemplate. The linear template DNA is obtained by contacting plasmid DNAwith a restriction enzyme under suitable conditions so that therestriction enzyme cuts the plasmid DNA at its recognition site(s) anddisrupts the circular plasmid structure. The plasmid DNA is preferablycut immediately after the end of the sequence which is to be transcribedinto RNA. Hence, the linear template DNA comprises a free 5′ end and afree 3′ end which are not linked to each other. If the plasmid DNAcontains only one recognition site for the restriction enzyme, thelinear template DNA has the same number of nucleotides as the plasmidDNA. If the plasmid DNA contains more than one recognition site for therestriction enzyme, the linear template DNA has a smaller number ofnucleotides than the plasmid DNA. The linear template DNA is then thefragment of the plasmid DNA which contains the elements necessary for invitro transcription, that is a promotor element for RNA transcriptionand the template DNA element. The open reading frame of the lineartemplate DNA determines the sequence of the transcribed RNA by the rulesof base-pairing.

In other embodiments, the nucleic acid template may be selected from asynthetic double stranded DNA construct, a single-stranded DNA templatewith a double-stranded DNA region comprising the promoter to which theRNA polymerase binds, a cyclic double-stranded DNA template withpromoter and terminator sequences or a linear DNA template amplified byPCR or isothermal amplification.

According to a preferred embodiment of the invention, the concentrationof the nucleic acid template comprised in the in vitro transcriptionmixture is in a range from about 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1to 20 nM, or about 1 to 10 nM. Even more preferred the concentration ofthe nucleic acid template is from about 10 to 30 nM. Most preferred theconcentration of the nucleic acid template is about 20 nM. In thiscontext it is particularly preferred to have a concentration of thenucleic acid template of about 1 to 200 μg/ml and more preferably ofabout 10 to 100 μg/ml, and most preferably of about 20 to 50 μg/ml (e.g.25 or 50 μg/ml).

RNA, mRNA: RNA is the usual abbreviation for ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotide monomers.These nucleotides are usually adenosine-monophosphate (AMP),uridine-monophosphate (UMP), guanosine-monophosphate (GMP) andcytidine-monophosphate (CMP) monomers or analogs thereof, which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificorder of the monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the RNA sequence. Usually RNA may beobtainable by transcription of a DNA sequence, e.g., inside a cell. Ineukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. In vivo, transcription of DNA usuallyresults in the so-called premature RNA which has to be processed intoso-called messenger-RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional-modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap,optionally a 5′UTR, an open reading frame, optionally a 3′UTR and apoly(A) sequence.

In addition to messenger RNA, several non-coding types of RNA existwhich may be involved in regulation of transcription and/or translation,and immunostimulation. The term “RNA” further encompasses RNA molecules,such as viral RNA, retroviral RNA and replicon RNA, small interferingRNA (siRNA), antisense RNA, CRISPR/Cas9 guide RNA, ribozymes, aptamers,riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA(rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA),microRNA (miRNA), and Piwi-interacting RNA (piRNA).

Dicarboxylic acid or salt thereof: A dicarboxylic acid is an organicacid having two carboxyl groups (—COOH). The term includes linearsaturated dicarboxylic acids having the general formulaHO₂C—(CH₂)_(n)—CO₂H such as oxalic acid, malonic acid, succinic acid,glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid andsebacic acid. It also includes unsaturated dicarboxylic acids having atleast one double bond such as maleic acid and fumaric acid as well assubstituted dicarboxylic acids having at least one additional functionalgroup such as malic acid, tartaric acid, cichoric acid anddimercaptosuccinic acid. The salt of the dicarboxylic acid comprises thedicarboxylic acid anion and a suitable cation such as Na⁺, K⁺, Ca²⁺ orMg²⁺.

Tricarboxylic acid or salt thereof: A tricarboxylic acid is an organicacid having three carboxyl groups (—COOH). Examples of tricarboxylicacids include citric acid, isocitric acid, aconitic acid, trimesic acid,nitrilotriacetic acid and propane-1,2,3-tricarboxylic acid. In thebuffer system and methods of the present invention preferably citricacid (3-carboxy-3-hydroxypentane-1,5-dioic acid) is used. The salt ofthe tricarboxylic acid comprises the tricarboxylic acid anion and asuitable cation such as Na⁺, K⁺, Ca²⁺ or Mg²⁺. Preferably, sodium ormagnesium citrate is used. If magnesium citrate is added to the RNA invitro transcription reaction, it is not necessary to add a magnesiumsalt to the reaction, since the magnesium ions within the magnesiumcitrate may serve as the cofactor for the RNA polymerase. Hence, in thiscase the reaction mixture for RNA in vitro transcription comprisesmagnesium citrate, a buffer substance, ribonucleoside triphosphates, anucleic acid template and RNA polymerase.

Buffer substance: A buffer substance is a weak acid or base used tomaintain the acidity (pH) of a solution near a chosen value after theaddition of another acid or base. Hence, the function of a buffersubstance is to prevent a rapid change in pH when acids or bases areadded to the solution. Suitable buffer substances for use in the presentinvention are Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) and HEPES(2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). The buffersubstance may further comprise an acid or a base for adjusting the pH,such as HCl in case of Tris (Tris-HCl) and KOH in case of HEPES(HEPES-KOH). In a preferred embodiment of the present invention citricacid is used to adjust the pH of the buffer substance, preferably ofTris base, so that no other acid has to be added. In an alternativeembodiment the pH of the buffer substance is adjusted with an acid or abase such as HCl and KOH and the salt of the dicarboxylic ortricarboxylic acid, preferably citrate, is present in the reactionmixture in addition to the pH-adjusted buffer substance.

The concentration of the buffer substance within the mixture for invitro transcription is 10 to 100 mM, 10 to 80 mM, 10 to 50 mM, 10 to 40mM, 10 to 30 mM or 10 to 20 mM. Preferably, the concentration of thebuffer substance is 80 mM.

Preferably the buffer has a pH value from 6 to 8.5, from 6.5 to 8.0,from 7.0 to 7.5, even more preferred of 7.5 or 8.0.

Ribonucleoside triphosphates: The ribonucleoside triphosphates (NTPs)GTP, ATP, CTP and UTP are the monomers which are polymerized during thein vitro transcription process. They may be provided with a monovalentor divalent cation as counterion. Preferably the monovalent cation isselected from the group consisting of Li⁺, Na⁺, K⁺, NH4⁺ ortris(hydroxymethyl)aminomethane (Tris). Preferably, the divalent cationis selected from the group consisting of Mg²⁺, Ba²⁺ and Mn²⁺. Morepreferably, the monovalent cation is Na ortris(hydroxymethyl)aminomethane (Tris).

According to a preferred embodiment of the invention, a part or all ofat least one ribonucleoside triphosphate in the in vitro transcriptionreaction mixture is replaced with a modified nucleoside triphosphate asdefined below.

Modified nucleoside triphosphate: The term “modified nucleosidetriphosphate” as used herein refers to chemical modifications comprisingbackbone modifications as well as sugar modifications or basemodifications. These modified nucleoside triphosphates are also termedherein as (nucleotide) analogs.

In this context, the modified nucleoside triphosphates as defined hereinare nucleotide analogs/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides are chemically modified. Asugar modification in connection with the present invention is achemical modification of the sugar of the nucleotides. Furthermore, abase modification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides. In this contextnucleotide analogs or modifications are preferably selected fromnucleotide analogs which are applicable for transcription and/ortranslation.

Sugar Modifications

The modified nucleosides and nucleotides, which may be used in thecontext of the present invention, can be modified in the sugar moiety.For example, the 2′ hydroxyl group (OH) can be modified or replaced witha number of different “oxy” or “deoxy” substituents. Examples of “oxy”-2′ hydroxyl group modifications include, but are not limited to, alkoxyor aryloxy (—OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroarylor sugar); polyethyleneglycols (PEG), —O(CH₂CH₂O)nCH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; and aminogroups (—O— amino, wherein the amino group, e.g., NRR, can bealkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) oraminoalkoxy.

“Deoxy” modifications include hydrogen, amino (e.g. NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified nucleotide can include nucleotidescontaining, for instance, arabinose as the sugar.

Backbone Modifications

The phosphate backbone may further be modified in the modifiednucleosides and nucleotides. The phosphate groups of the backbone can bemodified by replacing one or more of the oxygen atoms with a differentsubstituent. Further, the modified nucleosides and nucleotides caninclude the full replacement of an unmodified phosphate moiety with amodified phosphate as described herein. Examples of modified phosphategroups include, but are not limited to, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. The phosphate linker can also be modified by thereplacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

Base Modifications

The modified nucleosides and nucleotides, which may be used in thepresent invention, can further be modified in the nucleobase moiety.Examples of nucleobases found in RNA include, but are not limited to,adenine, guanine, cytosine and uracil. For example, the nucleosides andnucleotides described herein can be chemically modified on the majorgroove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In particularly preferred embodiments of the present invention, thenucleotide analogs/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

In some embodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

In other embodiments, modified nucleosides include 2-aminopurine,2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

In other embodiments, modified nucleosides include inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group.

In specific embodiments, a modified nucleoside is5′-O-(1-Thiophosphate)-Adenosine, 5′-O-(1-Thiophosphate)-Cytidine,5′-O-(1-Thiophosphate)-Guanosine, 5′-O-(1-Thiophosphate)-Uridine or5′-O-(1-Thiophosphate)-Pseudouridine.

In further specific embodiments the modified nucleotides includenucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine,8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine,2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-iso-cytidine,6-Chloro-purine, N6-methyl-adenosine, α-thio-adenosine,8-azido-adenosine, 7-deaza-adenosine.

Further modified nucleotides have been described previously (WO2013/052523).

Magnesium salt: A magnesium salt comprises a magnesium cation and asuitable anion such as a chloride or an acetate anion. Preferably, themagnesium salt is magnesium chloride. Preferably, the initial free Mg²⁺concentration is from about 1 to 100 mM, 1 to 75 mM, 1 to 50 mM, 1 to 25mM, or 1 to 10 mM. Even more preferred the initial free Mg²⁺concentration is from about 10 to 30 mM or about 15 to 25 mM. Mostpreferred is an initial free Mg²⁺ concentration of about 24 mM. Theperson skilled in the art will understand that the choice of the Mg²⁺concentration is influenced by the initial total NTP concentration,meaning that a higher Mg²⁺ concentration has to be used, if a highertotal NTP concentration is used in the in vitro transcription mixture.

RNA polymerase: The RNA polymerase is an enzyme which catalyzes thetranscription of a DNA template into RNA. Suitable RNA polymerases foruse in the present invention include T7, T3, SP6 and E. coli RNApolymerase. Preferably, a T7 RNA polymerase is used. Also preferably,the RNA polymerase for use in the present invention is a recombinant RNApolymerase, meaning that it is added to the RNA in vitro transcriptionreaction as a single component and not as part of a cell extract whichcontains other components in addition to the RNA polymerase. The skilledperson knows that the choice of the RNA polymerase depends on thepromoter present in the DNA template which has to be bound by thesuitable RNA polymerase. Preferably, the concentration of the RNApolymerase is from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about1 to 10 nM. Even more preferred, the concentration of the RNA polymeraseis from about 10 to 50 nM, 20 to 50 nM, or 30 to 50 nM. Most preferredis a RNA polymerase concentration of about 40 nM. In this context aconcentration of 500 to 10000 U/ml of the RNA polymerase is preferred.More preferred is a concentration of 1000 to 7500 U/ml and mostpreferred is a concentration of 2500 to 5000 Units/ml of the RNApolymerase. The person skilled in the art will understand that thechoice of the RNA polymerase concentration is influenced by theconcentration of the DNA template.

Pyrophosphatase: A pyrophosphatase is an acid anhydride hydrolase thathydrolyses diphosphate bonds. In the in vitro transcription reaction itserves to hydrolyze the bonds within the diphosphate released uponincorporation of the ribonucleoside triphosphates into the nascent RNAchain. Preferably, the concentration of the pyrophosphatase is fromabout 1 to 20 units/ml, 1 to 15 units/ml, 1 to 10 units/ml, 1 to 5units/ml, or 1 to 2.5 units/ml. Even more preferred the concentration ofthe pyrophosphatase is about 1 unit/ml or is about 5 units/ml.

5′-Cap structure: A 5′ cap is typically a modified nucleotide,particularly a guanine nucleotide, added to the 5′ end of an RNAmolecule. Preferably, the 5′ cap is added using a 5′-5′-triphosphatelinkage. A 5′ cap may be methylated, e.g. m7GpppN, wherein N is theterminal 5′ nucleotide of the nucleic acid carrying the 5′ cap,typically the 5′-end of an RNA. The naturally occurring 5′ cap ism7GpppN.

Further examples of 5′cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety.

Particularly preferred 5′ cap structures are CAP1 (methylation of theribose of the adjacent nucleotide of m7G), CAP2 (methylation of theribose of the 2^(nd) nucleotide downstream of the m7G), CAP3(methylation of the ribose of the 3^(rd) nucleotide downstream of them7G) and CAP4 (methylation of the ribose of the 4^(th) nucleotidedownstream of the m7G).

A 5′ cap structure may be formed by a cap analog.

Cap analog: A cap analog refers to a non-extendable di-nucleotide thathas cap functionality which means that it facilitates translation orlocalization, and/or prevents degradation of the RNA molecule whenincorporated at the 5′ end of the RNA molecule. Non-extendable meansthat the cap analog will be incorporated only at the 5′terminus becauseit does not have a 5′ triphosphate and therefore cannot be extended inthe 3′ direction by a template-dependent RNA polymerase.

Cap analogs include, but are not limited to, a chemical structureselected from the group consisting of m7GpppG, m7GpppA, m7GpppC;unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g.,m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylatedsymmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs(e.g., ARCA; m7,2′OmeGpppG, m7,2′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG andtheir tetraphosphate derivatives) (Stepinski et al., 2001. RNA7(10):1486-95). Examples of cap analogs are shown in Table 1.

TABLE 1 Cap analogs (D1 and D2 denote counterpart diastereoisomers)Triphosphate Tetraphosphate cap analog cap analog m⁷Gp₃G m⁷Gp₄G m₂^(7, 3’-O)Gp₃G b⁷Gp₄G b⁷Gp₃G b⁷m^(3’-O)Gp₄G e⁷Gp₃G m₂ ^(2, 7)Gp₄G m₂^(2, 7)Gp₃G m₃ ^(2, 2, 7)Gp₄G m₃ ^(2, 2, 7)Gp₃G b⁷m²Gp₄G m⁷Gp₃2’dGm7Gp⁴m⁷G m⁷Gp₃m^(2’-O)G m⁷Gp₃m⁷G m₂ ^(7, 2’-O)Gp₃G m₂ ^(7, 2’-O)GpppsG(D1) m₂ ^(7, 2’-O)GpppsG (D2) m₂ ^(7, 2’-O)GppspG (D1) m₂^(7, 2’-O)GppspG (D2) m₂ ^(7, 2’-O)GpsppG (D1) m₂ ^(7, 2’-O)GpsppG (D2)

Further cap analogs have been described previously (U.S. Pat. No.7,074,596, WO 2008/016473, WO 2008/157688, WO 2009/149253, WO2011/015347, and WO 2013/059475). The synthesis ofN⁷-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs has beendescribed recently (Kore et al., 2013. Bioorg. Med. Chem.21(15):4570-4).

Particularly preferred cap analogs are G[5′]ppp[5′]G, m⁷G[5′]ppp[5′]G,m₃ ^(2,2,7)G[5′]ppp[5′]G, m₂ ^(7,3′-O)G[5′]ppp[5′]G (3′-ARCA), m₂^(7,2′-O)GpppG (2′-ARCA), m₂ ^(7,2′-O)GppspG D1 (β-S-ARCA D1) and m₂^(7,2′-O)GppspG D2 (β-S-ARCA D2).

Preferably the cap analog is added with an initial concentration in therange of about 1 to 20 mM, 1 to 17.5 mM, 1 to 15 mM, 1 to 12.5 mM, 1 to10 mM, 1 to 7.5 mM, 1 to 5 mM or 1 to 2.5 mM. Even more preferred thecap analog is added with an initial concentration of about 5 to 20 mM,7.5 to 20 mM, 10 to 20 mM or 12.5 to 20 mM.

Ribonuclease inhibitor: A ribonuclease inhibitor inhibits the action ofa ribonuclease which degrades RNA. Preferably, the concentration of theribonuclease inhibitor is from about 1 to 500 units/ml, 1 to 400units/ml, 1 to 300 units/ml, 1 to 200 units/ml, or 1 to 100 units/ml.Even more preferred the concentration of the ribonuclease inhibitor isabout 200 units/ml.

Proteinogenic amino acid: A proteinogenic amino acid is an amino acidwhich is incorporated into protein during translation. Proteinogenicamino acids include alanine, serine, leucine, valine, isoleucine,glycine, histidine, proline, lysine, glutamic acid, glutamine, asparticacid, asparagine, arginine, selenocysteine, cysteine, tryptophan,methionine, phenylalanine, threonine and tyrosine.

Antioxidant: An antioxidant inhibits the oxidation of other molecules.Suitable antioxidants for use in the present invention include, but arenot limited to, DTT (dithiothreitol), TCEP(tris(2-carboxyethyl)phosphine), NAC (N-acetylcysteine),beta-mercaptoethanol, glutathione, cysteine and cystine. Preferably, DTTis used in the in vitro transcription reaction.

The concentration of the antioxidant, preferably of DTT, is 1 to 50 mM,5 to 48 mM, 8 to 47 mM, 10 to 46 mM, 15 to 45 mM, 18 to 44 mM, 20 to 43mM, 23 to 42 mM, 25 to 41 mM or 28 to 40 mM. Preferably, theconcentration is 40 mM.

Amine: Preferably, the amine to be used in the present invention isbetaine (trimethylglycine). The concentration of the amine, preferablyof betaine, may be 10 mM to 2M, preferably it is 0.7 M to 1.3 M.

Polyamine: Preferably, the polyamine is selected from the groupconsisting of spermine and spermidine. Preferably the concentration ofthe polyamine is from about 1 to 25 mM, 1 to 20 mM, 1 to 15 mM, 1 to 10mM, 1 to 5 mM, or about 1 to 2.5 mM. Even more preferred theconcentration of the polyamine is about 2 mM. Most preferred is aconcentration of 2 mM of spermidine.

DNase: DNases are enzymes which hydrolyze DNA by catalyzing thehydrolytic cleavage of phosphodiester linkages in the DNA backbone.Suitable DNases are isolated from bovine pancreas and are available fromvarious suppliers such as Sigma-Aldrich, New England Biolabs, Qiagen andThermoFisher. Preferably, the used DNase is free of any RNAse activity.In the method of the present invention the treatment with DNase isperformed after the RNA in vitro transcription reaction by adding theDNase to the reaction mixture used for RNA in vitro transcription.Preferably, a suitable amount of calcium chloride is added together withthe DNase to the RNA in vitro transcription mixture. The suitable amountof CaCl₂ is from 1 to 5 mM, preferably from 2 to 4 mM and morepreferably it is 3 mM. The DNA is treated with the DNase for 1 to 5hours, preferably for 1.5 to 3 hours and more preferably for 2 hours.The DNase treatment is preferably performed at a temperature of 37° C.In one embodiment, 3 mM CaCl₂ and 200 U/ml DNase I are added to the RNAin vitro transcription mixture and the resulting mixture is incubatedfor two hours at 37° C. In another embodiment, 3 mM CaCl₂ and 400 U/mlDNase I are added to the RNA in vitro transcription mixture and theresulting mixture is incubated for two hours at 37° C. The DNasetreatment can be stopped by adding EDTA or another chelating agent.Preferably, the DNase treatment is stopped by adding EDTA to a finalconcentration of 25 mM.

Bioreactor: The term bioreactor or transcription reactor as used hereinrefers to a chamber or test tube or column wherein an in vitrotranscription reaction is carried out under specified conditions. Thebioreactor may be thermally regulated to maintain accurately a specifictemperature, usually between 4 and 40° C. The bioreactor may beconfigured with an inflow or feed line and an exit port. The bioreactormay be a stirred-cell with provision for variable rates of stirring. Thebioreactor may comprise a filtration membrane for separating nucleotidesand other low molecular weight components from the reaction mixture.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, the present invention is based on the finding thatdicarboxylic or tricarboxylic acids reduce or prevent the formation ofprecipitates in a method of RNA in vitro transcription.

Hence, in a first aspect, the present invention relates to a method forin vitro transcription of a nucleic acid template into RNA, comprisingthe steps of:

-   -   providing a mixture comprising a dicarboxylic or tricarboxylic        acid or salt thereof, a buffer substance, ribonucleoside        triphosphates, one or more magnesium salts, said nucleic acid        template and RNA polymerase; and    -   incubating the reaction mixture under suitable conditions.

The skilled person knows that the concentration of the one or moremagnesium salt and the total concentration of ribonucleosidetriphosphates are related to each other, meaning that a higher amount ofthe magnesium salt has to be present, if a higher concentration ofribonucleoside triphosphates is used in the reaction. Generally, theconcentration of the magnesium salt should be slightly lower than thetotal concentration of ribonucleoside triphosphates. For example, if thetotal concentration of ribonucleoside triphosphates is 26.9 mM, theconcentration of the magnesium salt may be 24 mM.

In a similar way, the concentration of the dicarboxylic or tricarboxylicacid or salt thereof, preferably of citrate, may be adjusted to theconcentration of magnesium ions in the in vitro transcription reaction.Preferably, the concentration of the dicarboxylic or tricarboxylic acidor salt thereof, preferably of citrate, is at least half of theconcentration of magnesium ions within the in vitro transcriptionreaction. For example, the concentration of the dicarboxylic ortricarboxylic acid or salt thereof, preferably of citrate, has to be atleast 12 mM, if the concentration of magnesium ions is 24 mM. If a lowerconcentration of magnesium ions is used, the concentration of thedicarboxylic or tricarboxylic acid or salt thereof, preferably ofcitrate, can be reduced accordingly.

The concentration of the dicarboxylic or tricarboxylic acid or saltthereof, preferably of citrate, in the reaction mixture for in vitrotranscription may be in the range of 1 mM to 100 mM, of 3 mM to 80 mM,of 5 mM to 50 mM, of 8 to 40 mM, of 10 mM to 30 mM or of 12 mM to 25 mM.

In a preferred embodiment the reaction mixture contains 12 mM of thedicarboxylic or tricarboxylic acid or salt thereof, preferably ofcitrate, and 24 mM of magnesium ions, preferably of magnesium chloride.In another preferred embodiment the reaction mixture contains 24 mM of amagnesium salt of a dicarboxylic or tricarboxylic acid, preferably ofmagnesium citrate.

The method of the present invention can be operated in the batch mode,so that all reagents of the in vitro transcription reaction are presentin the reaction vessel from the beginning of the reaction and no newreagents are added and no product is removed before the in vitrotranscription reaction is stopped. Alternatively, the in vitrotranscription reaction may be operated in a semi-batch mode or in acontinuous mode. The term semi-batch as used herein refers to theoperation of the in vitro transcription reaction as a repetitive seriesof transcription reactions. For example, the reaction is allowed toproceed for a finite time at which point the product is removed, newreactants added, and the complete reaction repeated. The termcontinuous-flow as used herein refers to a reaction that is carried outcontinually in a bioreactor core with supplemental reactants constantlyadded through an input feed line and products constantly removed throughan exit port.

The skilled person knows that in a reaction operated in a semi-batchmode or in a continuous mode the initial concentration of ribonucleosidetriphosphates and magnesium can be lower than in the batch mode, ifadditional amounts of these reagents are added at some time after thestart of the in vitro transcription reaction. In this case, theconcentration of the dicarboxylic acid or tricarboxylic acid or saltthereof can also be lower than in the batch mode.

The total concentration of all four ribonucleoside triphosphates in thereaction mixture is 0.1 to 60 mM, 1 to 50 mM, 3 to 40 mM, 8 to 35 mM, 10to 30 mM or 12 to 25 mM. If the in vitro transcription reaction isperformed in the semi-batch or continuous mode, the initial totalconcentration of the four ribonucleoside triphosphates in the reactionmixture is 0.1 to 10 mM, 0.5 to 8 mM or 1 to 7 mM. In this case, theinitial concentration of magnesium ions in the reaction mixture is 0.1to 8 mM, 0.5 to 7 mM or 1 to 6 mM.

In the in vitro transcription reaction mixture the ribonucleosidetriphosphates can all be present in the same amount. For example, 6 mMATP, 6 mM UTP, 6 mM CTP and 6 mM GTP can be used in the in vitrotranscription reaction mixture. Alternatively, a sequence-optimizedreaction mixture can be used, wherein the fraction of each of the fourribonucleoside triphosphates within the reaction mixture corresponds tothe fraction of the respective nucleotide in the RNA molecule which isto be produced by the method. Such a sequence-optimized reaction mixtureis disclosed in PCT/EP2015/001164. The present invention can also beused to produce homopolymer RNAs, i.e. RNAs consisting of only one typeof nucleotides. In this case, the concentration of the ribonucleosidetriphosphate which forms the homopolymer is the same as the totalconcentration of ribonucleoside triphosphates.

The RNA produced by the in vitro transcription can be purified by anysuitable method, including DNA template digest, phenol-chloroformextraction, LiCl precipitation, HPLC and/or tangential flow filtration.Preferably, the RNA is purified by HPLC using a porous reversed phase asstationary phase. Such a process is described in WO 2008/077592. Alsopreferably, the RNA is purified by tangential flow filtration. Such aprocess is described in PCT/EP2015/062002.

As discussed before, the addition of a dicarboxylic or tricarboxylicacid or salt thereof, preferably of citrate, to an in vitrotranscription reaction reduces or prevents the formation of precipitatesduring the in vitro transcription process. The formation of precipitatescan be detected spectrophotometrically by measuring the absorption of asample taken from the in vitro transcription reaction at a wavelength of350 to 550 nm, preferably at a wavelength of 500. The absorption at 500nm increases when precipitates form. A suitable apparatus for detectingthe precipitates is Nanodrop 2000 UV spectrophotometer. Alternatively,the precipitates can be separated from the soluble compounds and then beanalyzed, for example by centrifugation, size exclusion chromatography,dynamic or static light scattering, automated particle sorting orultracentrifuge fractionation.

The addition of a dicarboxylic or tricarboxylic acid or salt thereof,preferably of citrate, to an in vitro transcription reaction reduces theformation of precipitates during the in vitro transcription processcompared to an in vitro transcription reaction with the same reactionmixture which does not contain said dicarboxylic or tricarboxylic acidor salt thereof by at least 10% or 20%, preferably by at least 30%, 40%or 50%, more preferably by at least 60%, 70% or 80%, even morepreferably by at least 90%, 95% or 98% and most preferably noprecipitates can be detected in an in vitro transcription reactioncontaining said dicarboxylic or tricarboxylic acid or salt thereof.

In another aspect, the present invention relates to the use of an amine,preferably of betaine, in the RNA in vitro transcription of a nucleicacid template with a high percentage of guanosine and cytosinenucleotides.

A betaine is a neutral chemical compound with a positively chargedcationic functional group such as a quaternary ammonium or phosphoniumcation which bears no hydrogen atom and with a negatively chargedfunctional group such as a carboxylate group which may not be adjacentto the cationic site. Preferably, the betaine is trimethylglycine, i.e.the amino acid glycine which has three methyl groups bound to thenitrogen atom of the amino acid.

A nucleic acid template is considered to have a high percentage ofguanosine and cytosine nucleotides, if the number of guanosine andcytosine nucleotides is at least 50%, preferably at least 55% and morepreferably at least 60% of the total number of nucleotides within anucleic acid template. The high percentage of guanosine and cytosinenucleotides may be present in the complete nucleic acid template or apart thereof which has a length of at least 10% or 20%, preferably of atleast 30% or 40%, more preferably of at least 50% or 60% and even morepreferably of at least 70% or 80% of the total length of the nucleicacid template.

Without wishing to be bound by the theory, it is hypothesized that theuse of betaine, preferably of trimethylglycine, in the RNA in vitrotranscription of a nucleic acid template with a high percentage ofguanosine and cytosine nucleotides reduces the formation of secondarystructures which may reduce the transcription efficiency.

Accordingly, the present invention also relates to the use of betaine,preferably of trimethylglycine, in the reduction of secondary structuresin a nucleic acid template for RNA in vitro transcription having a highpercentage of guanosine and cytosine nucleotides.

EXAMPLES

The following examples are intended to illustrate the invention in afurther way. They are merely illustrative and not intended to limit thesubject matter of the invention.

Example 1 Preparation of DNA and mRNA Constructs

For the present examples, a DNA sequence encoding Photinus pyralisluciferase (PpLuc) was prepared by modifying the wild type encodingPpLuc DNA sequence by GC-optimization for stabilization (SEQ ID NO: 1).The GC-optimized PpLuc DNA sequence was introduced into a pUC19 derivedvector and modified to comprise a alpha-globin-3′-UTR (muag (mutatedalpha-globin-3′-UTR)), a histone-stem-loop structure, and a stretch of70×adenosine at the 3′-terminal end (poly-A-tail). The obtained plasmidDNA was used for RNA in vitro transcription experiments (see Example 4and 5) to obtain PpLuc RNA (SEQ ID NO: 2).

Example 2 Characterization of Precipitates Formed During RNA In VitroTranscription Using a State-of-the-Art HEPES-KOH Buffer

The goal of that experiment was to characterize the composition ofprecipitates that are formed during RNA in vitro transcription.

RNA In Vitro Transcription and Analysis of Precipitates:

The RNA in vitro transcription reaction was performed using a linear DNAtemplate as described in Example 1. The reaction mixture also contained24 mM MgCl₂, 13.45 mM NTP mixture, 16.1 mM Cap analog and 2500 units/mlT7 RNA Polymerase. As buffer system HEPES-KOH pH 8.0 was used. Atcertain time points of the transcription reaction, samples were taken (0h, 1 h, 2 h, 3 h, 4 h). Both precipitates that accumulated in thereaction tube and the supernatant of the reaction were further analyzedon a conventional SDS-polyacrylamide gel. The result of this analysis isshown in FIG. 1.

Results:

The results show that the precipitates formed during RNA in vitrotranscription (reaction buffer HEPES-KOH) contain RNA and proteins.

Example 3 Preparation of RNA In Vitro Transcription Buffers

In the following, the preparation of the conventional RNA in vitrotranscription buffers (HEPES and Tris-HCl) and RNA in vitrotranscription buffers comprising citrate or acetate is described.

Preparation of the HEPES Buffer:

First, a 5× stock HEPES buffer was prepared, comprising 400 mM HEPES, 10mM spermidine, 200 mM DTT and 25 units/ml pyrophosphatase. The pH of thebuffer was set to 7.5, using KOH. The HEPES buffer was used in 1×concentration in the RNA in vitro transcription experiment (see Example4).

Preparation of Tris Buffers:

First, a 5× stock TRIS buffer was prepared, comprising 400 mM Tris, 10mM spermidine, 200 mM DTT and 25 units/ml pyrophosphatase. To prepare aTris-HCl buffer pH 7.5, the titration of the pH was done with HCl. Toprepare a Tris-acetate buffer pH 7.5, the titration of the pH was donewith acetic acid. To prepare a Tris-citrate buffer pH 7.5, the titrationof the pH was done with 4 M citric acid. In addition, all prepared TRISbuffers also contained 25 mM KOH. KOH was added to enable a bettercomparability of the obtained results, because the HEPES buffer alsocontained KOH (see Example 4). The Tris buffers (Tris-HCl buffer pH 7.5,Tris-acetate buffer pH 7.5, Tris-citrate buffer pH 7.5) were used in 1×concentration in the RNA in vitro transcription experiment (see Example4).

Example 4 RNA In Vitro Transcription Using Different Buffer Systems

The goal of this experiment was to characterize the effect of differentRNA in vitro transcription buffers on the formation of precipitates. Inthat experiment, buffers known in the art (HEPES, Tris-HCl) and bufferscomprising acetate or citrate (Tris-Acetate, Tris-Citrate) were tested.The RNA in vitro transcription reaction was performed over 90 minutes,and three different samples taken at 30 minutes, 60 minutes and 90minutes were analyzed spectrometrically (absorption at 500 nm) to detectprecipitates.

RNA In Vitro Transcription Reaction:

The RNA in vitro transcription reaction was performed using a linear DNAtemplate as described in Example 1. The reaction mixture also contained24 mM MgCl₂, 13.45 mM NTP mixture, 16.1 mM Cap analog and 2500 units/mlT7 RNA Polymerase. As buffer systems, either HEPES-KOH pH 7.5, Tris-HClpH 7.5, Tris-acetate pH 7.5, or Tris-citrate pH 7.5 were used (preparedaccording to Example 3). Samples of the respective reactions were takenat 0 minutes, 30 minutes, 60 minutes and 90 minutes to monitor theformation of precipitations.

Spectrometric Detection of Precipitates Formed During the RNA In VitroTranscription:

The samples obtained during the RNA in vitro transcription reactionswere spectrometrically analyzed (Nanodrop 2000 UV-spectrometer). Theabsorption of at a wavelength of 500 nm is a measure to determine thepresence of precipitates. The results of the measurements are shown inFIG. 2.

Results:

The results show that the use of a Tris buffer prepared with themonocarboxylic acid acetic acid (Tris-acetate buffer pH 7.5) led to theformation of more precipitates during RNA in vitro transcriptioncompared to the state-of-the-art buffers HEPES and Tris-HCl. However,the results surprisingly show that the Tris-buffer prepared with citricacid (Tris-citrate, pH 7.5) completely blocked the formation ofprecipitates (see FIG. 2).

Example 5 RNA In Vitro Transcription Using Tris Buffers with DifferentCitrate Concentrations

The goal of this experiment was to characterize the effect of citrateobserved in Example 4 in more detail. Therefore, buffers with differentcitrate concentrations ranging from 0 mM citrate to 25 mM citrate wereprepared, and the effect on precipitate formation was analyzed. The RNAin vitro transcription reaction was performed over 70 minutes, and twodifferent samples taken at 30 minutes and 70 minutes werespectrometrically analyzed (absorption at 500 nm) to detectprecipitates.

RNA In Vitro Transcription Reaction and Detection of Precipitates:

The RNA in vitro transcription reaction mixture was prepared asdescribed in Example 4. The RNA in vitro transcription reaction wasperformed in eight different Tris-HCl buffers (pH 7.95) containingdifferent concentrations of citrate (0 mM-25 mM sodium citrate). At 30minutes and 70 minutes, samples were taken and spectrometricallyanalyzed (according to Example 4). The results of the experiment areshown in FIG. 3.

Results:

The results show that the reduction of precipitations was adose-dependent effect of citrate. Moreover, the experiment showed thatat citrate concentrations >10 mM, the formation of precipitates wasalready strongly reduced, and at concentrations >15 mM, the formation ofprecipitates could be completely prevented (see FIG. 3).

Example 6 RNA In Vitro Transcription Using HEPES Buffers with DifferentCitrate Concentrations

The goal of this experiment was to evaluate if the observed effect ofcitrate on the prevention of precipitates in Tris buffer is alsotransferrable to other buffers such as HEPES.

Therefore, HEPES-KOH buffers pH 8.0 with different citrateconcentrations ranging from 0 mM citrate to 22.45 mM citrate (i.e. 0 mM;13.45 mM; 17.95 mM; 22.45 mM citrate) were prepared, and the effect onprecipitate formation was analyzed. The RNA in vitro transcriptionreaction was performed over 120 minutes, and samples for each buffercondition were taken at 120 minutes and analyzed spectrometrically(absorption at 550 nm) to detect precipitates. The results are shown inFIG. 4.

Results:

The results show that the reduction of precipitations in aHEPES-buffered reaction was a dose-dependent effect of citrate.Moreover, the experiment showed that at citrate concentrations >10 mM,the formation of precipitates was already strongly reduced, and atconcentrations >15 mM, the formation of precipitates could be completelyprevented (see FIG. 4).

Example 7 DNA Hydrolysis After RNA In Vitro Transcription

The goal of this experiment was to evaluate whether the observedpositive effect on the prevention of precipitates also improves andoptimizes the hydrolysis of the DNA template after RNA in vitrotranscription. DNA template is conventionally hydrolyzed using DNAses,enzymes that need Ca²⁺ ions and Mg²⁺ ions to be active and efficient. Anideal RNA in vitro transcription buffer should promote the function ofDNAses to hydrolyze DNA template, eventually preventing DNAcontaminations of the final RNA product.

DNA Digestion of the Template:

RNA in vitro transcription was essentially performed according toExample 1, using four different buffer systems (Tris-HCl; Tris-HCl+6 mMCitrate; Tris-HCl+10 mM Citrate; HEPES-KOH+10 mM Citrate).

In a first set of experiments, DNA template was removed by adding 3 mMCaCl₂ and 200 U/ml DNAse1 (Thermo Fisher) and incubating for 2 h at 37°C. The digestion reaction was stopped by adding EDTA to a finalconcentration of 25 mM.

In a second set of experiments, DNA template was removed by adding 3 mMCaCl₂ and 400 U/ml DNAse1 (Thermo Fisher) and incubating for 2 h at 37°C. The digestion reaction was stopped by adding EDTA to a finalconcentration of 25 mM.

Analysis of Residual pDNA Using qPCR:

A qPCR method was used to detect residual pDNA using specific primersand probes for the origin of replication of the pDNA. Quantitative PCRwas performed using a LightCycler and LightCycler Master Mix (RocheDiagnostics) according to the manufacturer's instructions. The resultsof the analysis are shown in FIG. 5.

Results:

The results show that the inventive RNA in vitro transcription buffersystem comprising citrate promotes the hydrolysis of the DNA template byDNase (see FIG. 5).

The inventive buffer system is therefore suitable to produce RNA withreduced DNA contaminations. Moreover, the more efficient DNA hydrolysismay also economize the industrial RNA production process, as less DNAseenzyme is needed to digest pDNA template.

1. Method for in vitro transcription of a nucleic acid template intoRNA, comprising the steps of: providing a mixture comprising adicarboxylic or tricarboxylic acid or salt thereof, a buffer substance,ribonucleoside triphosphates, one or more magnesium salts, said nucleicacid template and a recombinant RNA polymerase, wherein the mixture doesnot comprise a proteinogenic amino acid or tRNA; and incubating thereaction mixture under suitable conditions.
 2. Method for preparing RNA,comprising the steps of: a) incubating a mixture comprising adicarboxylic or tricarboxylic acid or salt thereof, a buffer substance,ribonucleoside triphosphates, one or more magnesium salts, a nucleicacid template and a recombinant RNA polymerase under suitableconditions; b) adding a DNase to the mixture of a) and incubating theresulting mixture containing DNase under suitable conditions.
 3. Methodaccording to claim 1 or 2, wherein the dicarboxylic or tricarboxylicacid or salt thereof is citric acid or a salt thereof.
 4. Methodaccording to claim 3, wherein the concentration of citric acid or saltthereof is at least half of the concentration of magnesium ions presentin the in vitro transcription reaction.
 5. Method according to any oneof the preceding claims, wherein the buffer substance is Tris base,HEPES or Tris-HCl.
 6. Method according to any one of the precedingclaims, wherein the concentration of the buffer substance is 10 to 100mM.
 7. Method according to any one of the preceding claims, wherein thedicarboxylic or tricarboxylic acid or salt thereof is used to adjust thepH of the mixture.
 8. Method according to any one of the precedingclaims, wherein the reaction mixture comprises Tris-citrate or HEPES-KOHplus sodium citrate, ribonucleoside triphosphates, one or more magnesiumsalts, said nucleic acid template and RNA polymerase.
 9. Methodaccording to any one of the preceding claims, wherein the magnesium saltis magnesium chloride.
 10. Method according to claim 9, wherein theconcentration of magnesium chloride is 1 to 100 mM.
 11. Method accordingto any one of the preceding claims, wherein the reaction mixture has apH of 6 to 8.5.
 12. Method according to any one of the preceding claims,wherein the reaction mixture has a pH of 7.5 to 8.0.
 13. Methodaccording to any one of the preceding claims, wherein the RNA polymeraseis T7 RNA polymerase.
 14. Method according to any one of the precedingclaims, wherein the total concentration of ribonucleoside triphosphatesin the mixture is between 0.1 and 60 mM.
 15. Method according to any oneof the preceding claims, wherein the nucleic acid template is alinearized plasmid DNA template.
 16. Method according to any one of thepreceding claims, wherein the reaction mixture further comprises one ormore of ribonuclease inhibitor, pyrophosphatase, cap analog, one or moreantioxidants and one or more amines and/or polyamines.
 17. Methodaccording to claim 16, wherein the antioxidant is DTT.
 18. Methodaccording to claim 17, wherein the concentration of DTT is 1 to 50 mM.19. Method according to claim 18, wherein the polyamine is spermidine.20. Method according to claim 19, wherein the concentration ofspermidine is 1 to 25 mM.
 21. Method according to any one of claims 2 to20, wherein the mixture in b) further comprises a calcium salt. 22.Method according to claim 21, wherein the calcium salt is calciumchloride.
 23. Method according to claim 22, wherein the concentration ofcalcium chloride in the mixture is 3 mM.
 24. Method according to any oneof the preceding claims, further comprising a step (c) of purifying theRNA.
 25. Method according to claim 24, wherein the step of purifying theRNA comprises HPLC using a porous reversed phase as stationary phaseand/or tangential flow filtration.
 26. Buffer for in vitro transcriptionof RNA comprising a dicarboxylic or tricarboxylic acid or a salt thereofand Tris as a buffer substance.
 27. Buffer according to claim 26,wherein the dicarboxylic or tricarboxylic acid or a salt thereof iscitric acid or citrate.
 28. Buffer according to claim 27, wherein theconcentration of citric acid or citrate is at least 1 mM.
 29. Bufferaccording to any one of claims 26 to 28, having a pH of 6 to 8.5. 30.Buffer according to any one of claims 26 to 29, being Tris-citrate. 31.Reaction mixture for in vitro transcription comprising a dicarboxylic ortricarboxylic acid or a salt thereof, a buffer substance, ribonucleosidetriphosphates, one or more magnesium salts, a nucleic acid template anda recombinant RNA polymerase, but not comprising a proteinogenic aminoacid or tRNA.
 32. Reaction mixture according to claim 31, furthercomprising one or more of ribonuclease inhibitor, pyrophosphatase, capanalog, one or more antioxidants and one or more amines and/orpolyamines.
 33. Reaction mixture according to claim 31 or 32, whereinthe dicarboxylic or tricarboxylic acid or a salt thereof is citric acidor citrate.
 34. Reaction mixture according to claim 33, wherein theconcentration of citric acid or citrate is at least 1 mM.
 35. Reactionmixture according to any one of claims 31 to 34, wherein the buffersubstance is Tris or HEPES.
 36. Reaction mixture according to any one ofclaims 31 to 35, having a pH of 6 to 8.5.
 37. Reaction mixture accordingto any one of claims 31 to 36, wherein the buffer substance isTris-citrate or HEPES-KOH plus sodium citrate.
 38. Kit for in vitrotranscription, comprising the buffer according to any one of claims 26to 30 or the reaction mixture according to any one of claims 31 to 37.39. Bioreactor for in vitro transcription, comprising the bufferaccording to any one of claims 26 to 30 or the reaction mixtureaccording to any one of claims 31 to
 37. 40. Use of a dicarboxylic ortricarboxylic acid or a salt thereof in the reduction or prevention ofthe formation of precipitates in a method of in vitro transcription. 41.Use according to claim 40, wherein the method of in vitro transcriptionis as defined in any one of claims 1 or 3 to
 25. 42. Use of adicarboxylic or tricarboxylic acid or a salt thereof in promoting thehydrolysis of DNA by DNase.
 43. Use according to claim 42, wherein theDNA hydrolysis takes place after RNA in vitro transcription.